In humans, P. aeruginosa infection is a cause of significant mortality in cystic fibrosis patients. In P. aeruginosa there are 3 or 4 distinct signaling pathways that utilize conserved chemotaxis proteins to independently control flagella, twitching and biofilm development. The mechanism by which crosstalk is prevented in these similar yet functionally distinct complexes is unknown, but likely involves segregation of these complexes to different cellular locations. I have shown that both transmembrane methyl-accepting chemotaxis proteins (MCPs) and one soluble MCP, McpS, are localized to the cell poles. This finding is surprising and reveals that the spatial organization of the different chemotaxis systems in P. aeruginosa is different than that of other characterized bacteria. My immediate goal is to identify factors essential for the polar localization of the soluble MCPs. Toward this end, in aim 1, I will identify the cognate chemotaxis system and the localization determinants for the soluble MCPs (McpL and McpS). Because the mechanism of polar clustering of soluble MCPs is unknown, we will address whether they exist in higher order complexes that require a cognate CheW, a critical coupling protein for chemotaxis clustering in other bacteria. Should I find that the polar clustering of the soluble MCPs is through a heretofore unexplored mechanism, I will identify the polarity factors responsible for their localization. Twitching motility is meditated by type IV pili, and is controlled by the Pil/Chp operon. Virtually nothing is known about the intracellular location of this signaling pathway nor are all of the proteins involved known. In aim 2, I will identify the core components of the signaling pathway and define the rules that govern their localization. Finally, in E. coli, methylation is critical for the clustering of some, but not all, MCPs. Moreover, the methyltransferase only binds to those MCPs with a C-terminal pentapeptide sequence. The importance of methylation on MCP clustering and the mechanism by which the methyltransferase binds to MCPs is almost completely unexplored in other bacteria. Thus, in aim 3, I will characterize the chemotaxis methylation mechanism and its role in MCP localization. Completion of these studies will lay the foundation for my long-term goal of determining how spatial location is achieved and how it plays a role in P. aeruginosa virulence.